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Category Archives: Genetic Engineering

Agrobacterium is probably one of the most famous and oldest genetic engineers. It injects a marked piece of its “Ti-Plasmid” into plant cells. The inserted sequence codes for a growth hormone (to make the plant produce this “tumors”, but it does not have much to do with cancer. Just unregulated growth of these plant cells) and a gene to make the plant produce a special nutrient, that many other bacteria cannot digest. Thus it programs the plant to give it shelter and food. Smart bug.

For the GFP plant, a lab strain of this bacterium was taken. The marked piece of DNA, that it usually injects into plants was deleted (disarmed strain) and later replaced with the GFP gene. So the only gene it could possibly inject is the sequence we provide it with in pGreenII.

Here’s an actual map of pVIB that shows the Insert (from Vibrio Fischeri genomic DNA) between the two SalI sites. It was inserted into the SalI site from pBR322, disrupting the Tetracyclin resistance gene. BglII cuts within the luxI coding site (in the green line), so the Ribosome binding site of LuxC is still intact. Thus by restiction with SalI and BglII you get a functional LuxCDABEG casette. Just attach a constitutive promoter before it (BglII), to get constitutive expression. Or a chloroplast promoter, to get glowing chloroplasts. Only one thing is annoying – there is a transcription terminator directly after LuxG (between LuxG and SalI site). No restriction site inbetween to cut away the terminator.

The green fragment (~1700 bp) contains a cell density induced Promoter, and the LuxI gene, and LuxR in the opposite direction. The native Lux cassette constantly produces the LuxR protein, and small amonuts of LuxI (which is an enzyme that produces the autoinducer). The autoinducer diffuses throgh the cell wall into the medium, and to some extent also in other cells. At high cell densities, much autoinducer is in both the medium and in the cell. The autoinducer binds to LuxR protein which then activates strong expression (1000-fold the constitutive expression) of the LuxICDABEG operon. (LuxI is then also expressed at higher levels).

Someday I will retransform pVIB and then add myristic acid to the petri dish. That should enhance the glow significantly. pVIB on steroids 😀

I heard that some microbiologists for routine plasmid amplification just put a colony of E.Coli into LB in an Eppi, add plasmid, incubate it for 30 minutes and then place on selective medium. Transformation efficiency is very bad, so not good for ligation!

Had to try that out today… Let’s see what will happen 😀

So I did three tries:

One in H2O with 0,9% NaCl 37°C, LB 37°C and one in LB 20°C…

Just inocculated E.Coli with a pipette tip into a sterile eppendorf tube, with 200 uL medium/water. Added a GFP-coding plasmid… Waited for 30 Minutes and plated them on LB-Apmicillin-Agar…

Tomorrow we’ll see if colonies were formed and if they glow under UV-Light also.

As a result, these plates were obtained…

With a bacterial lawn growing on the plates… Seems the Ampicillin of those (months old) plates has decayed too much… Guess there will be a chance to do it again soon…

Wouldn’t it be awsome to walk in a park in the late evening, under bioluminescent trees? Or have a bonsai in your room that glows? I just adore bioluminescence, especially in plants!!

At the moment I’m rewriting the lux genes of a bacterium to make them fit in a plant.

The biggest burden, however, is money. Having the DNA synthesized will cost around 3200-3500 Dollars (depending on promoter lenght and price per base pair). Don’t forget the costs of an agrobacterium strain including plasmids (60$) and the restriction enzyme (either for free from university, or 57$ per 5000 units, no smaller package available). But all in all, these costs are neglible.

EDIT: Restriction enzymes will not be needed at all, because the company will be ordered to clone the synthesized gene into a Ti-plasmid.

So I’ll just be designing the gene sequence, and while doing so looking for a way to get it funded anyhow. Maybe there’s a science competition, a prize, etc. I think kickstarter.com is just for US citizens… Have to look closely at that again…

It surely can be realized, because plants can produce any enzyme needed and the aldehydes.

After getting a protocol from the DIY bio mailing list how to do a miniprep without EDTA* mostly using home chemicals (http://wiki.biohackers.la/Miniprep ) I try to get Diatomaceous Earth.

It would be really cool to be able to kind of ‘produce’ my own plasmid solutions. For always having some in stock. And for trying to transform bacteria other than E.Coli, without having to buy new plasmids (9$ plasmid costs + 35$ shipping from US).

*(In former times EDTA was used for developing photos from negatives (film strips). Technically, you can order EDTA for those purposes on the web. But, like always, shipment costs are quite high compared to the value of the substances. I still regret one day at university we did a titration with EDTA and then disposed of it. Should have taken it at home, would have been free… 50-100 mL from each group (7 groups) would have been a lot of stuff to me)

EDIT: Did a miniprep at university using only those chemicals I got. Yield was ~276 ng/uL, and 260:280 was also a fair value.

In short, I inserted the genes for bioluminescence (of a marine bacterium) into E. Coli @university.

If you want to learn more about transformation, google “heat shock transformation”. There is plenty to find on google. And make sure to check out “How to do a pGLO Transformation” (by biscuitpiggy2) on youtube. It clearly shows the mechanism of DNA uptake using plasmids.

EDIT:

I once re-streaked a frozen culture on very fresh LB-amp plates. It was amazing! One could see the glowing in a not-very-dark room, even though the streetlights was shining in. Probably because the ampicillin worked better, and because the inoculated bacteria were from a luminescent colony, there were much more bacteria carrying the plasmids than from the first “generation” – thus glowing way brighter.